The term “DNA cloning” generally refers to the technique of binding a gene fragment to a vector having the ability to self-replicate, such as a plasmid, phage, or cosmid, introducing the product into a host such as Escherichia coli (E. coli), and causing the host to proliferate to produce the same gene cluster. Cloning and subcloning in E. coli is conducted by the method of binding a target gene fragment that has been amplified by a method such as polymerase chain reaction (PCR) with a vector having a replication origin and an antibiotic selective marker by using DNA ligase and introducing the product into E. coli cells. Subsequently, the cloned bacteria can be sorted by checking for resistance to the antibiotic.
Existing DNA cloning methods are limited by the fact that the DNA that is inserted and the vector must be cleaved with a restriction enzyme recognizing the same site in both, the fact that a restriction enzyme must be selected that does not cleave the interior of the inserted DNA or the vector, and the like.
In recent years, homologous recombination techniques and site-specific recombination techniques employing sequence-specific genetically modified enzymes that promote the recombination of DNA fragments by recognizing specific base sequences have been employed in the cloning of target gene fragments. They have also begun to be widely used to rapidly clone large quantities of genes and express proteins without processing with restriction enzymes or the like.
However, even when employing homologous recombination techniques, the steps of amplifying plasmids with microorganisms such as E. coli and purifying are still necessary. The operation is still tedious and lacks economy.
The joint polymerase chain reaction (jPCR) is known as a method of producing gene clusters having an identical base sequence by joining a target gene fragment with one or more DNA fragments having a specific function other than the target gene under conditions permitting the expression of the function (see Patent Reference 1 and Non-patent References 1 and 2). Here, the term “DNA fragments having a specific function other than the target gene” means a promoter sequence and a polyadenylation signal.
In the method described in Patent Reference 1, three DNA fragments independently containing a promoter sequence, target gene sequence, and polyadenylation signal are PCR amplified with RNA-DNA chimera primers. The amplified DNA fragments are processed with RNase to remove the RNA primer sites. The complementarity of the 3′ protruding regions produced on the termini of the individual DNA fragments is utilized to combine the three DNA fragments using DNA ligase. PCR conducted using this product as a template then permits the production of a large quantity of joined DNA in which the three DNA fragments are functionally joined in the order of promoter sequence, target gene sequence, and polyadenylation signal (see Patent Reference 1).
Non-patent References 1 and 2 describe a method in which a promoter sequence, target gene sequence, and polyadenylation signal are independently amplified by using primers which have been produced by adding a sequence homologous with the terminal region of the DNA fragment that is to be joined to the 5′ end of the primers used in gene amplification. By conducting jPCR with these three DNA fragments, it is then possible to produce joined DNA fragments in which the promoter sequence, target gene sequence, and polyadenylation signal are functionally joined (see FIG. 2 in Non-patent Reference 1 and FIG. 1 in Non-patent Reference 2).
The target gene fragment is normally prepared as a PCR product. However, when the binding specificity of the primers to the template is low in PCR, when multiple sequences similar to the primer sequences are present in the template, and the like, nonspecific amplification reactions take place. As a result, regions that are sandwiched with primer sequences at either end will end up producing nonspecifically amplified DNA fragments that are not in the target gene being cloned.
In conventional jPCR, sequences for joining are added to the primers used to amplify the various DNA fragments. A sequence hybridizing with a different DNA fragment is introduced onto the terminus of each DNA fragment being amplified with a given primer. Next, the amplified DNA fragments are mixed and jPCR is conducted to form “joined DNA fragments” in which two or more DNA fragments are bound.
However, in this method, fragments consisting of regions sandwiched between primer sequences in the PCR product obtained by the gene amplification reaction with sequences not derived from the target gene also form “joined DNA fragments” in the same manner as target gene fragments.
Even assuming that just target gene fragments are amplified by the gene amplification reaction, the primers employed in gene amplification remain in the reaction solution. When jPCR is conducted without removing them, the amplification of various DNA fragments that are constituent elements of the “joined DNA fragments” takes place preferentially over the amplification of the “joined DNA fragments,” and “joined DNA fragments” containing sequences derived from the desired target gene tend not to form.
Accordingly, in the methods described in the above-cited patent reference and non-patent references, the PCR amplification product must be purified to a degree where it is possible to ignore the effects of contamination by DNA fragments containing non-target gene sequences and primers following PCR amplification of the target gene fragments.
As set forth above, to produce a “joined DNA fragment” combining two or more double-stranded DNA fragments by the above method, the DNA fragments containing the target gene sequence that are contained in the PCR amplification product must be purified to a degree where the effects on the joining reaction of contamination by DNA fragments containing non-target gene sequences and the primers used in target gene amplification can be ignored in a step conducted prior to joining one or more DNA fragments having specific functions other that of the target gene. However, when the length of the target gene fragment is similar to the length of a non-target gene fragment, separation of the target gene fragment is difficult. Thus, in such cases, it becomes impossible to obtain just “joined DNA fragments” containing the sequence derived from the desired target gene.
Accordingly, the object of the present invention is to provide a method permitting the specific production of a “joined DNA fragment” containing a sequence derived from a desired target gene by causing one or more double-stranded DNA fragments to bind to a PCR amplification product containing a target gene sequence without purifying the PCR amplification product.